Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method

ABSTRACT

The invention relates to a polypeptide for the enzymatic detoxification of zearalenone, said polypeptide being a monooxygenase which converts the keto group in position 7 of zearalenone into an ester group, the monooxygenase in particular being an amino acid sequence selected from the group comprising sequence ID No. 1, 2 and 3 or a functional variant thereof. The functional variant and at least one of the amino acid sequences has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80% and most preferably 90%,

BACKGROUND OF THE INVENTION

The present invention relates to a polypeptide for the enzymatic detoxification of zearalenone, a transgenic host cell for the preparation of a zearalenone-detoxifying monoxygenase, an additive for the enzymatic detoxification of zearalenone, and the use of an isolated polynucleotide, and a method for preparing a zearalenone-detoxifying monoxygenase.

Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative of the same is the widespread zearalenone (ZEN), formerly known as F-2 toxin, which is produced by a plurality of Fusarium fungi. These fungi inter alia infest cultivated plants such as various cereal crops, fungal infestation usually occurring prior to the harvest, wherein the growth of the fungi, or production of mycotoxins my also occur prior to the harvest or, with improper storage, even after the harvest, FAQ estimates that, on a global scale, 25% of the agrarian products are contaminated with mycotoxins, which results in considerable economic losses. In a study recently performed worldwide, a total of 23,781 samples were analyzed from January 2009 to 2011, 81% having been tested positive for at least one mycotoxin and 45% having been tested positive for ZEN, ZEN was found at a frequency of up to 100% in all regions of the world and in all tested cereal and feed classes such as maize, soy meal, wheat, wheat bran, DDGS (distillers dried grains with solubles) as well as in finished animal feed mixtures.

ZEN is a non-steroidal, estrogenic, macrocyclic lactone synthesized via the polyketide metabolic pathway and having the structural formula:

and the IUPAC nomenclature naming (2E, 11D) -15,17-dihydroxy-11-methyl-12-oxabicyco[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione. There are different nomenclatures for ZEN and ZEN metabolites, the nomenclature of Metzler (2011, Mycotox. Res., 27:1-3) having been adopted in this document as far as possible. Besides ZEN, a plurality of ZEN derivatives formed by enzymatic or chemical modifications of ZEN also occur in nature. Furthermore, ZEN metabolites are inter alia formed in the human or animal organism.

ZEN as well as ZEN derivatives such as α-ZEL or β-ZEL are also detectable in processed food or feed products, such as bread, beer or DDGS, due to their high chemical and physical stability.

Although ZEN comprises a relatively low acute toxicity and an oral LD50 value of up to 20,000 mg/kg body weight, subacute and/or subchronic toxic effects such as teratogenic, carcinogenic, immunosuppressive and estrogenic effects may occur in animals or humans after extended intake. ZEN binds to the estrogen receptor and may cause hormonal disorders, and hence a reduction of the formed metabolites as compared to ZEN is usually understood by detoxification of zearalenone.

The intake of feeds contaminated with ZEN results in development disorders in mammals, pigs and, in particular, young animals, being extremely sensitive to ZEN. ZEN concentrations above 0.5 ppm in feeds will lead to development disorders, wherein, for instance, concentrations above 1.5 ppm may lead to hyper-estrogenicity in pigs, and concentrations of 12 ppm have been made responsible for bovine miscarriages. Since zearalenone is rapidly absorbed via mucous membranes, in particular via the mucous membranes of the stomach, yet also of the mouth, an immediate and, above all, quantitative detoxification is necessary. Already 30 minutes after the oral administration of ZEN, the latter is detectable in blood. In order to achieve as complete a detoxification as possible, the use of isolated enzymes was found to be advantageous over microorganisms, the former exhibiting a higher specific activity or more rapid action. Due to the harmful effect of ZEN, the European Union established binding upper limits of ZEN in foods and gave recommendations for ZEN upper limits in feeds (EC NO: 1881/2006).

The primary strategy for reducing ZEN contamination in foods or feeds is to restrict the growth of fungi, for instance by observing “good agricultural practices”. These includes seeds free of pests and fungal infestation, or the removal of agricultural waste products from fields in due time. Furthermore, the growth of fungi in the fields can be reduced by the use of fungicides. After the harvest, the crop should be stored at a residual moisture of below 15% and a low temperature in order to prevent the growth of fungi. Similarly, items contaminated by fungal infestation should be removed prior to further processing. Despite this list of measures, I. Rodriqes and K. Naehrer reported (2012) that even in regions with the highest agricultural standards, such as the USA and Central Europe, 29% and 39%, respectively, of the tested maize samples were contaminated with ZEN from 2009 to 2011.

Other options for removing ZEN from feeds or foods comprise the adsorption or the transformation of the mycotoxin. To this end, it is necessary that the binding of the mycotoxin to the adsorbent is strong and specific over a wide pH range and remains stable in the entire gastrointestinal area throughout the digestive process. Although some non-biological adsorbents such as activated carbon, silicates or synthetic polymers like cholestryamine can be efficiently used for aflatoxins, their use for other mycotoxins is limited. The essential disadvantage of adsorbents is the non-specific binding of other molecules partially essential for nutrition. Biological adsorbents such as yeast or yeast extracts are also described in the literature, yet are similarly limited as non-biological adsorbents.

The detoxification of ZEN by physical and chemical treatments is also limited. ZEN cannot be effectively deactivated by thermal treatment, yet the ZEN content can be reduced by 83.9% by extrusion and treatment with oxidants, e.g. for 16 hours at 80° C. with a 10% hydrogen peroxide solution. The use of extrusion methods and oxidants like ozone or hydrogen peroxide in the production of feeds and foods is limited due to the high costs, loss of quality, sometimes low efficiency and low specificity involved.

DESCRIPTION OF RELATED ART

From EP 0 938 575 B1, ZEN-degrading properties of bacteria of the genus Rhodococcus and Nocardia, in particular R. globerulus, R. erythropolis and N. globerula, are known. Vekiru et al. (Appl. and Environ. Microb., 2010, 76, 7, 2353-2359) describes the biotransformation of ZEN to the less estrogenic metabolite ZOM-1 by the microorganisms Trichosporon mycotoxinivorans.

The terms used below are taken from the technical terminology and, unless otherwise stated, are each used in their conventional meanings. Thus, the term “polynucleotide” refers to any kind of genetic material of any length and sequence, such as single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural elements, groups of genes, plasmids, entire genomes and fractions thereof. The term “polypeptide” comprises proteins such as enzymes, antibodies and polypeptides with up to 500 amino acids, such as peptide inhibitors, domains of proteins, yet even short polypeptides with small sequence lengths, e.g. less than 10 amino acids, such as receptors, ligands, peptide hormones, tags and the like. The term “position” in a polynucleotide or polypeptide refers to a single, specific base or amino acid in the sequence of said polynucleotide or polypeptide, respectively.

BRIEF SUMMARY OF THE INVENTION

The present invention aims to provide a polypeptide that enables the rapid and reliable conversion of zearalenone to a zearalenone derivative whose toxicity and estrogenic effect are reduced to such an extent as to be able to remain in feeds or food products while being harmless to the respective end users.

To solve this object, the present invention is essentially characterized in that the polypeptide is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and that the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2 and 3 or a functional variant thereof, the functional variant and at least one of the amino acid sequences having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%. Monooxygenases are enzymes belonging to group I of the EC classification, which catalyze the incorporation of an oxygen atom from O₂ into the respective substrate, i.e. zearalenone in the present case. More particularly, in the case of zearalenone, an oxygen atom is incorporated in position 7 of the ring structure of zearalenone, thus obtaining a less strongly toxic zearalenone derivative (iZOM). Surprisingly, it has been found that such detoxification occurs in just a single enzymatic reaction step, and that the reaction product iZOM has a toxicity that is lower by a factor of about 100, and virtually no longer exhibits an estrogenic effect, despite the closed ring structure still present and the carbon oxygen double bond in position 7.

A particularly complete conversion of zearalenone into the less toxic derivative iZOM will be possible if the polypeptide is an amino acid sequence selected from the group consisting of SEQ ID1, 2 and 3 or a functional variant thereof, the functional variant and at least one of the amino acid sequences having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%. Due to the presence of at least one such conserved amino acid sequence, such as the sequences of ID Nos. 1, 2 and 3, or a functional variant thereof, it is possible to provide a polypeptide which, in addition to the rapid and complete conversion of ZEN, also comprises a particularly high activity value as compared to hitherto known ZEN-transforming polypeptides.

It has so far been assumed that a cleavage of the lactone ring system is required for an efficient detoxification of ZEN. It has, however, been surprisingly found that an effective detoxification will also be achieved by the conversion of the keto group in position 7 of zearalenone to an ester group, during which rearrangement no cleavage of the ring system takes place.

According to a further development of the invention, the monooxygenase is selected such that it is a Baeyer-Villiger monooxygenase. The use of a Baeyer-Villiger monooxygenase ensures that the reaction will exclusively occur at the desired position 7, since this is the only site accessible for a so-called Baeyer-Villiger oxidation, i.e. the conversion of a ketone to an ester by incorporating an oxygen atom.

According to a further development of the invention, the polypeptide is selected such that the polypeptide, in particular the monooxygenase, converts zearalenone to iZOM in a one-step enzymatic oxidation reaction so as to detoxify at least 70% of the zearalenone within 24 hours at 30° C. by converting the keto group in position 7 of the zearalenone into an ester group, said polypeptide being formed by a transformed yeast strain YZGA515, which is transformed with a pCS57 vector additionally containing a polynucleotide for expressing the polypeptide, and the transformed yeast strain is used at a cell density OD600 of 4, and zearalenone is used as a substrate at a concentration of 2 mg/l, and SC-LEU medium is used as a reaction medium. This is, in particular, feasible because the reaction product iZOM surprisingly has a toxicity reduced by a factor of about 100.

Due to the specific structure of the zearalenone molecule and, in particular, due to the keto group present in position 7 of the zearalenone molecule, the selective incorporation of the desired oxygen atom in position 7 of the zearalenone by a subgroup consisting of the Baeyer-Villiger monooxygenase, namely cyclohexanone monooxygenase, has become possible, as in correspondence with a further development of the invention. The ester group formed by such incorporation in a surprising manner causes the formed cyclic metabolite (iZOM) to have a significantly reduced toxicity as compared to zearalenone.

By the term “sequence identity”, a percental identity is understood. For amino acid sequences and nucleotide sequences, the relative sequence identity can be determined visually, yet preferably calculated by a computer program. The amino acid sequences of SEQ ID Nos. 1, 2 and 3, and the nucleotide sequences of SEQ ID Nos. 3, 4 and 5 are defined as reference sequences. A sequence comparison is also performed within sequence sections, a section meaning a continuous sequence of the reference sequence. Normally, the length of the sequence sections for nucleotide sequences is 18 to 600, preferably 45 to 200, more preferably 100 to 150, nucleotides. Normally, the length of the sequence sections for peptide sequences is 3 to 200, more preferably 15 to 65, most preferably 30 to 50, amino acids. There is a plurality of purchasable or costlessly available bioinformatic programs that can be used for the determination of homology and are constantly updated. Examples include GCG Wisconsin Bestfit package (Devereux et al. 1984), BLAST (Altschul et al. 1990) or BLAST 2 (Tatusova und Madden 1999). Due to different adjustment options of these algorithms, it may happen that different results are output at identical input sequences, which is why such algorithms and the associated adjustments have to be defined. In the present case, the sequence identity was performed using the programs NCBI BLAST (Basic Local Alignment Search Tool), in particular BLASTP for polypeptides and BLASTN for polynucleotides, in the versions of Oct. 20, 2014, which are available on the website of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). It is thereby possible to compare two or several sequences with one another according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25: 3389-3402). In this case, the programs versions of May 15, 2013, were used. As program adjustments, the base adjustments were applied, yet, in particular, for the amino acid comparison: “max target sequence”=100; “expected threshold”=10; “word size”=3; “matrix”=BLOSOM62; “gap costs”=“Existence: 11; Extension: 1”; “computational adjustment”=“Conditional compositional score matrix adjustment”; and for the nucleotide sequence comparison: Word Size: 11; Expect value: 10; Gap costs: Existence=5, Extension=2; Filter=low complexity activated; Match/Mismatch Scores: 2, −3; Filter String: L; m.

The terms “functional polypeptide variant” or “functional variant”, on the one hand, refer to “allelic variants” of the polypeptide and to “functional fragments” of the polypeptide and, on the other hand, refer to a “modified polypeptide”, wherein the enzymatic function is substantially unchanged as compared to the polypeptide of SEQ ID No.1. The term “allelic variant” refers to a polypeptide that was formed by nucleotide sequence mutation(s) accidentally occurring in nature and causes a change in the amino acid sequence, wherein the enzymatic function is unaffected thereby. The term “functional fragment” relates to a part or partial sequence of a polypeptide, or a part or partial sequence of a functional variant thereof, wherein the enzymatic function is substantially maintained. “Modified polypeptides” may comprise C- or N-terminal fusion proteins or selectively mutated polypeptides, mutations being obtainable by the substitution, insertion or deletion of at least one amino acid and, in particular, by site-specific mutagenesis or accidental mutagenesis, recombination and/or any other protein-engineering method, wherein the enzymatic function is substantially maintained. The terms substitution, insertion and deletion are used in the meanings common in genetic engineering and familiar to the skilled artisan. An enzymatic function is substantially maintained if the enzymatic reaction mechanism remains unchanged, i.e. the keto group in position 7 of the mycotoxin ZEN is converted to a corresponding ester group as shown in the above-mentioned reaction mechanism, and the specific residual activity is at least 5%, preferably at least 10%, in particular at least 50%, based on the original polypeptide.

Polypeptides having amino acid sequences SEQ ID Nos. 1 and 3 are functional allelic variants, with the sequences each derived from different microorganisms. This is clearly apparent from the close relative relationship, measured by the percentual sequence identity, and from the fact that the polypeptides act on ZEN by the same mechanism. The polypeptide of SEQ ID No. 1 is completely contained in the sequence SEQ ID No. 2, yet the polypeptide of SEQ ID No. 2 comprises an N-terminus extended by 28 amino acids.

By the terms “detoxification” or “detoxify”, a reduction of the estrogenic activity of ZEN is understood. The measurement of the estrogenic activity is preferably performed according to the method described in the exemplary embodiments. Other methods may, however, also be applied, the decisive factor always being a reduction of the estrogenic activity of ZEN by its enzymatic conversion to iZOM. Measured by the estrogenicity assay described in the exemplary embodiments, iZOM has an estrogenic activity lower than that of ZEN by a factor of about 100.

Moreover, an isolated polynucleotide can be provided, by which it is possible to prepare a polypeptide for the rapid and reliable detoxification of ZEN. To this end, the isolated polynucleotide has a nucleotide sequence encoding a polypeptide that is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and/or has a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6. In this case, the nucleotide sequence is able to hybridize, under medium stringency conditions, with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4 to 6 and/or a partial sequence thereof of at least 200 nucleotides, in particular at least 100 nucleotides, and/or a complementary strand of the nucleotide sequence or partial sequence thereof. The expression of such a polynucleotide ensures that the resulting polypeptide transforms ZEN into iZOM.

Nucleotide sequences to be expressed, in particular their triplets (codons), are usually changed as a function of their host cell such that the codon bias is optimized as a function of the host cell. This results in that even polynucleotides having a degree of sequence identity of far below 80%, yet even below 70% or below 60%, will be able to encode one and the same polypeptide. A sequence comparison for determining the degree of sequence identity must also be performed within sequence sections, a section meaning a continuous sequence of the reference sequence. Normally, the length of sequence sections for nucleotide sequences is 15 to 600.

By the aid of the present isolated nucleotide sequences or sequence sections, it has become possible to generate nucleic acid probes having lengths of usually at least 15, 30 or 40nucleotides. Such nucleotide sequences, which are referred to as probes and in most cases are additionally labeled, e.g. with ³H, ³²P, ³⁵S, biotin or avidin, can be identified by applying standard method nucleotide sequences encoding polypeptides with ZEN-degrading activity. DNA, RNA or cDNA of individual microorganisms, genomic DNA libraries or cDNA libraries can, for instance, be used as starting materials for the identification of such sequences.

For nucleotide sequences having lengths of at least 100 nucleotides, medium stringency conditions are defined as prehybridization or hybridization at 42° C. in a five-fold NaCl-containing Na-EDTA buffer (SSPE, 0.9 M NaCl, 60 mM NaH₂PO₄, 6 mM EDTA) containing 0.3% sodium dodecyl sulfate (SDS), 200 μg/ml sheared and denaturated salmon sperm DNA and 35% formamide, followed by standard Southern Blot conditions, wherein the carrier material is finally washed three times with double sodium chloride citrate buffer (SSC, 300 mM NaCl und 30 mM trisodium citrate, 0.2% SDS) at 55° C. for 15 minutes.

For nucleotide sequences having lengths of 15 to 100 nucleotides, medium stringency conditions are defined as prehybridization or hybridization in a buffer comprising 0.9 M NaCl, 0.09 M Tris-HCl pH=7.6, 6 mM EDTA, 0.5% NP-40, single Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate, 0.1 mM ATP and 0.2 mg/ml yeast RNA, wherein said prehybridization and hybridization are effected at temperatures of 5° C. to 10° C. below the calculated melting temperatures (Tm), Tm being determined by calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390). After this, the test is continued under standard Southern Blot conditions (J. Sambrook, E. F. Fritsen and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition. Cold Spring Harbor, New York). The carrier material is finally washed once with a six-fold sodium chloride citrate buffer containing 0.1% SDS for 15 minutes and twice with a six-fold SSC buffer, each for 15 minutes at 5° C. to 10° C. below the calculated Tm.

Another object of the invention is to provide a transgenic host cell for preparing a zearalenone-detoxifying monoxygenase.

To solve this object, the invention is essentially characterized in that the host cell expresses a polynucleotide, that the polynucleotide comprises a nucleotide sequence encoding at least one polypeptide according to any one of claims 1 to 4 and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, said polynucleotide being chromosomally integrated or extrachromosomally present and the host cell being a plant cell, and that optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively.

An enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, and cofactors required therefor can be taken from the prior art, for instance from Torres Pazmino et al. 2008 (Angew. Chem. 47(12); 2275-8). Enzymes converting NADP+ or NAD+ to NADPH or NADH, respectively, are preferably selected so as to be usable without any problems in the food and/or feed industries and, in particular, comply with the respective food and/or feed regulations. Thus, it is, for instance, possible to use an NADPH-dependent xylose reductase, in particular from Pichia stipitis, which uses as substrate xylitol, which is admitted as feed additive, as said enzyme converting NADP+ to NADPH. Alternatively, an NAD(P)H-converting mannitol dehydrogenase from Lactobacillus sp. can be used, wherein mannitol, which is admitted as feed additive, can be used as substrate.

In a preferred manner, it is possible to prepare, or express, as fusion protein the polypeptide for detoxifying ZEN and an enzyme capable of recycling the cofactor. It is thereby possible to provide the two enzymatic activities in a joint process, in particular a biotechnological upstream and downstream process. When applying such a fusion protein, it is, moreover, ensured that, the cofactor circulating between the two oxidation steps remains in spatial proximity to the polypeptide for detoxifying ZEN.

If the host cell is a plant cell, such a transgenic host cell enables the production of seed, comprising said transgenic plant cell.

Finally, the present invention aims to provide an additive that enables the rapid and reliable detoxification, defined as reduction of the estrogenic activity, of ZEN in a defined or complex matrix, such as, for instance, animal feeds or food products.

To solve this object, a zearalenone-detoxifying additive is provided, which additive comprises at least one polypeptide being a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, which monooxygenase preferably comprises an amino acid sequence selected from SEQ ID Nos. 1, 2 and 3 or a functional variant thereof, the functional variant and at least one of the amino acid sequences having a sequence identity of at least 60%, and optionally at least one adjuvant selected from the group consisting of vitamins, minerals, enzymes, further components for detoxifying mycotoxins and cofactors, in particular NADPH and/or NADH, enzyme preparations such as proteases, amylases, cellulases or glucanases, hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases and/or combinations thereof, mycotoxin-detoxifying enzymes such as aflatoxin oxidase, ergotamine hydrolases, ergotamine amidases, ochratoxin amidases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol aminoxidases, deoxynivalenol epoxide hydrolases; mycotoxin-detoxifying microorganisms; mycotoxin-binding components such as microbial cell walls, and inorganic materials such as bentonite. Such an additive in a food or feed product directly enables the one-step biochemical conversion of ZEN to a zearalenone derivative, namely iZOM, the toxicity of which is lower by a factor of about 100 than that of zearalenone. By using such an additive, it will be ensured that the major portion of the amounts of ZEN contained, for instance, in food, or feed products will most certainly be oxidized to iZOM, thus preventing the occurrence of any harmful effect on the organism of a subject receiving such food or feed products.

The additive, in addition to a polypeptide according to the invention, may, for instance, also comprise as adjuvant an enzyme preparation in which at least one enzyme is further contained, which, for instance, participates in the degradation of proteins, e.g. proteases, or which participates in the metabolism of starch or fiber or fat or glycogen, e.g. amylases, cellulases or glucanases, as well as, for instance, hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases and/or combinations thereof.

Further possible adjuvants usable according to the invention include enzyme preparations that, in addition to at least one polypeptide according to the invention, contain at least one component for detoxifying mycotoxins different from zearalenone, such as a mycotoxin-detoxifying enzyme like aflatoxin oxidase, ergotamine hydrolases, ergotamine amidases, ochratoxin amidases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol aminoxidases, deoxynivalenol epoxide hydrolases; and/or at least one mycotoxin-detoxifying microorganism and/or at least one mycotoxin-binding component e.g. microbial cell walls, or inorganic materials such as bentonite.

Furthermore, the polypeptide is contained in the adjuvant in a concentration enabling the rapid, conversion of ZEN to the non-toxic or significantly less toxic metabolite iZOM, in particular already prior to its resorption by the body of a subject, in particular mammal, eating contaminated feed or food.

The polypeptide may be present in the composition in encapsulated or coated form, wherein standard methods such as described, for instance, in WO 92/12645 can be used for encapsulating or coating. Such encapsulation or coating enables the polypeptide to be transported to its place of use without change, in particular degradation or damage, such that the polypeptide will not start acting before the dissolution of its protective coat, e.g. in the digestive tract of animals, thus achieving an even more selective, rapid and complete transformation of ZEN even in acid, protease-rich and anaerobic environments. By such encapsulation or coating, it is, moreover, also possible to enhance the temperature stability of the polypeptide in the additive.

The encapsulated or coated polypeptide can be further processed to premixes using adjuvants common in the feed industry.

The invention. further relates to the use of an isolated polynucleotide encoding at least one polypeptide according to the invention and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 for preparing a zearalenone-detoxifying monooxygenase, and to the use of an isolated polynucleotide encoding at least one polypeptide according to the invention and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 in a method for preparing a zearalenone-detoxifying monooxygenase.

The present invention, moreover, relates to the use of the additive for detoxifying zearalenone in feed products, in particular for pigs, poultry and aquaculture; in food products; or in distillers dried grains with solubles. The use according to the invention of the additive enables the ZEN contained in the food or feed product or DDGS to be oxidized to iZOM, and hence detoxified, by converting the keto group in position 7 into an ester group, such detoxification being already feasible at low polypeptide concentrations in contaminated feed or food products.

The amount of polypeptide per ton of feed or food or DDGS lies within a range from about 1 g to about 500 g, the encapsulated or coated polypeptide ranging from about 20 g to about 300 g, the premix ranging from about 200 g to about 10 kg, or the additive ranges from about 5 mg to about 10 kg. This enables the ZEN contained in feed or food products, which may be present in concentrations up to 10,000 μg/kg, to be converted to iZOM for the major portion.

If the additive is used in starch liquefaction processes, in saccharification processes, in biogas processes, or in fermentation processes such as the mashing or fermentation process in the production of bioethanol, it will be safeguarded that ZEN contained in the products used for the processes or resulting therefrom, such as DDGS or starch, will be converted to iZOM such that no health-damaging amounts of ZEN will remain intact.

The present invention further aims to provide a method for preparing a zearalenone-detoxifying monooxygenase that enables the rapid and reliable conversion of ZEN to a non-toxic derivative, namely iZOM.

To solve this object, the method is conducted such that an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to any one of claims 1 to 4 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into a transgenic host cell or extrachromosomally provided, that the host cell is a prokaryotic or eukaryotic cell, in particular a yeast or a plant cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell. Such an isolated polynucleotide encoding a zearalenone-detoxifying polypeptide can further be used for preparing a host cell in such a manner that the host cell expresses the isolated polynucleotide, which comprises a nucleotide sequence encoding at least one polypeptide according to any one of claims 1 to 4 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, wherein the polynucleotide is chromosomally integrated or extrachromosomally provided and the host cell is a prokaryotic or eukaryotic cell, in particular a yeast or a plant cell, and optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively.

In that the zearalenone is converted to iZOM, and hence detoxified, by the addition of a polypeptide, in particular a monooxygenase, in a one-step enzymatic oxidation reaction, wherein the keto group in position 7 of the zearalenone is converted into an ester group, and that optionally a cofactor oxidized in the reaction, in particular NADP+ or NAD+, is reduced to NADPH or NADH, respectively, by a further enzyme, not only the rapid incorporation of the oxygen atom forming the ester group together with the keto group in position 7 will be guaranteed, but it will above all be ensured that the reaction will run to completion and zearalenone will be completely converted to iZOM, which is less toxic by a factor of about 100. NADP+ or NAD+ can be converted or recycled to NADPH or NADH, respectively, by the already described enzymatic systems.

In this case, a polypeptide having an amino acid sequence selected from. SEQ ID Nos. 1, 2 and 3, or a functional variant thereof, can be used as polypeptide for the enzymatic detoxification of zearalenone, wherein the functional variant and at least one of the amino acid sequences have a sequence identity of at least 60%. When using a polypeptide having an amino acid sequence selected from SEQ ID Nos. 1, 2 and 3, a particularly complete detoxification of zearalenone has been observed.

The method according to the invention can be applied both in an additive per se and in a transgenic host cell, a transgenic plant, or in seeds, and regardless of the place of use will lead to consistently good results and, in particular, a complete transformation of zearalenone to iZOM.

In this case, the method can be conducted such that the polypeptide, or the additive, is mixed with a feed or food product contaminated with zearalenone, the obtained mixture is contacted with moisture, and the polypeptide, or the additive, detoxifies the zearalenone contained in the contaminated feed or food product. With moist feed or food products, such as mash or pulp, the conversion of zearalenone to iZOM takes place in the feed or food product prior to its oral intake so as to ensure that the harmful effects of zearalenone on humans and animals will be largely eliminated. By moisture, the presence of water or a water-containing liquid is understood, this also encompassing, for instance, saliva or other liquids present in the digestive tract.

The method according to the invention can also be conducted such that the feed or food product is pelletized prior to its oral intake.

According to a further development of the invention, the method for preparing a transgenic host cell is conducted such that an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to any one of claims 1 to 4 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host, cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell.

Similarly, the invention relates to the use of a transgenic host cell for preparing a zearalenone-detoxifying monooxygenase, which is characterized in that an isolated, polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to any one of claims 1 to 4 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host cell, and that, optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following, the invention will be explained in more detail by way of exemplary embodiments and drawings. Therein,

FIG. 1 depicts the partial transformation of ZEN in the negative control by S. cerevisiae;

FIG. 2 depicts the transformation of ZEN in the test setup by S. cerevisiae, which expresses the polypeptide of SEQ ID No. 1;

FIG. 3 illustrates the chemical structure of iZOM; and

FIG. 4 shows the estrogenic activity of ZEN (FIG. 4A) and of iZOM (FIG. 4B).

DETAILED DESCRIPTION OF THE INVENTION

Unless indicated in more detail, all molecular-biological and microbiological operations were performed using standard techniques (Methods in Enzymology, Volume 134, Pages 3-933 (1991); Guide to Yeast Genetics and Molecular Biology, Edited by Christine Guthrie, Gerald R. Fink. ISBN: 978-0-12-182095-4; J. Sambrook et al. 2012, Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor).

EXAMPLE 1 Formation of iZOM From ZEN in a One-Step Reaction

The yeast strain YZGA515 (Saccharomyces cerevisiae modified by inactivation of the ABC transporter gene PDR5 (Poppenberger et al. 2003, J. Biol. Chem., 278(48)47905-14) for the enhanced absorption of zearalenone (ZEN)) was transformed by the plasmid pCS57 using standard techniques. pCS57 is a plasmid derived from the yeast expression plasmid pADH-FW (Mitterbauer et. al. 2002, Appl. Environ. Microbiol.; 68(3), 1336-1346) with LEU2 as selection marker and ADH1 promoter.

In the test setup, the yeast strain YZGA515 was transformed with the pCS57 vector additionally containing the polynucleotide of SEQ ID No. 4 as BamHI-Xhol fragment after the strongly constitutive ADH1 promoter (alcohol dehydrogenase 1). This enabled the formation of the polypeptide of SEQ ID No. 1.

The yeast strain YZGA515 with the empty vector (pADH-FW without the polynucleotide of SEQ ID No.4) was transformed as negative control.

The transgenic yeasts (test setup and negative control) were cultivated in SC-LEU medium (Sherman 1991, Methods Enzymol., 194, 3-21), centrifuged and resuspended in fresh SC-LEU medium at a concentration of 2 mg/l ZEN, wherein a cell density of OD600=4 was adjusted in both cases. After various incubation times, samples were taken, supplemented with 1 volume methanol, and the intracellular processes were thus stopped. The cell suspension was cleared by centrifugation, and the resulting supernatants were used for HPLC-MS measurements. The measurements, as described above, comprised the starting substance zearalenone (ZEN) and the transformation product ZOM-1formed by Trichosporon mycotoxinivorans (Vekuri et al. 2010, Appl. Environ. Microbiol. 76(7)2353-9).

In the negative control transformed with the empty vector, no ZOM-1 and, in particular, no iZOM was formed. The respective HPLC analysis data are illustrated in FIG. 1, the y-axis indicating nanogram per milliliter (ng/ml) and the x-axis indicating the time in hours (h).

By contrast, zearalenone was significantly more rapidly transformed in the test setup, and a new metabolite with the expected mass of the intermediate postulated by Vekiru et al. (2010) and referred to as iZOM below was identified. In addition, ZOM-1 was also detectable, which clearly suggested that a hydrolysis of iZOM to ZOM had occurred to some extent. The respective HPLC analysis data are illustrated in FIG. 2. Compared to the negative control, a much smaller amount of ZEN was measured, the y-axis indicating nanogram per milliliter (ng/ml) and the x-axis indicating the time in hours (h).

In a further test, setup, the metabolite iZOM was additionally measured quantitatively. Table 1 shows the molar balance of the metabolization of ZEN. From this, it is clearly apparent that the major portion of the used zearalenone (ZEN) was already converted to iZOM after 6 hours. The small shortfall in the balance is explainable by the formation of further metabolites and the method-related analytical unsharpnesses. The results clearly show that the polypeptide of SEQ ID No. 1 is able to convert ZEN to its metabolite iZOM.

TABLE 1 Molar balance of the metabolization of ZEN in test Setup Time ZEN ZOM-1 iZOM [h] [nM] [nM] [nM] 0 3364 0 0 6 172.1 0 2163.1 24 4.8 214.8 2068.2

EXAMPLE 2 Identification of the Chemical Structure of iZOM

The chemical structure of the metabolite iZOM (FIG. 3) could be determined by NMR measurements of the isolated and purified metabolite. For measuring the NMR spectra, a sufficient amount of iZOM was prepared from several liters of the culture filtrate of a yeast culture (SC-LEU as described in Example 1). iZOM was purified by solid phase extraction and subsequent preparative HPLC.

The use of the isolated reference substance for iZOM on the one hand allowed for the tracing of the conversion of ZEN to iZOM (cf. Example 1) and, on the other hand, enabled the clarification of the chemical structure by NMR.

The NMR spectra for identifying iZOM were obtained in a CD3OD solution using a Bruker Avance DRX-400 FT NMR spectrometer at room temperature (20° C.) with a 5 mm inverse broadband z-gradient probe head. The chemical displacements were established based on the residual solvent resonance (3.31 ppm for ¹H NMR, 49.15 ppm for ¹³C NMR). All pulse programs were taken from the Bruker software library. The NMR data were evaluated by means of TopSpin 1.3 (Bruker BioSpin GmbH). The determination of the complete structure and the assignment of signals were performed based on ¹H, ¹³C-APT, ¹H²H correlation spectroscopy (COSY), ¹H¹³C heteronuclear single quantum correlation spectra (HSQC), and ¹H¹³C heteronuclear multiband correlation spectra (HMBC).

The NMR spectroscopy data of iZOM show two essential differences as compared to ZEN. These confirm the conversion of the keto group to an ester group. The differences substantially comprise the shift of the carbonyl carbon atom from 212 ppm in ZEN (typical of a ketone) to 175 ppm (typical of a carboxylic acid derivative) and the shift of the adjacent CH₂ group from 36 ppm and 2.90/2.30 ppm (position 8 in ZEN) to 65 ppm and 4.20/4.05(position 9 in iZOM) for ¹³C and ¹H, respectively, caused by the adjacent oxygen atom. Unlike the open ring of ZOM1, the macrocyclic ring in iZOM is still intact as evidenced by a long-range correlation between C7 and H9. The NMR data are indicated in Table 2. The chemical structure of iZOM is illustrated in FIG. 3.

TABLE 2 ¹H and ¹³C NMR date for identifying iZOM ¹H ¹³C Position δ (ppm) Multiplicity, J (Hz) δ (ppm)  1 — 172.9  3 5.14 m 74.2  3-CH₃ 1.36 d, 6.2 20.2  4 1.65-1.80 m 36.5  5 1.90, 1.76 m 23.1  6 2.30-2.45 m 35.8  7 — 175.4  9 4.18, 4.05 m 64.6 10 1.75-1.90 m 28.9 11 2.30-2.50 m 30.8 12 5.93 ddd, 15.4, 8.3, 6.1 132.4 13 7.00 d, 15.4 133.2 14 6.40 d, 2.2 109.6 15 — 165.0 16 6.19 d, 2.2 103.3 17 — 165.6 18 — 104.9 19 — 143.9

EXAMPLE 3 Strongly Reduced Estrogenic Activity of iZOM as Compared to ZEN

The purified metabolite iZOM as described in Example 2 was used to determine its toxicity, expressed by its estrogenic activity, and compared to that of zearalenone.

For measuring the estrogenic activity, a specially prepared reporter yeast strain, YZHB817 (Bachmann, H.: Phenotypic detection of zearalenone in Saccharomyces cerevisiae. Master thesis BOKU Vienna, 2003) was used. This strain is derived from yeast-two hybrid strain PJ69-4a (MATa trpl-901 leu2-3,112 ura3-52 his3-200 gal4- (deleted) gal80 (deleted) LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lac2) (James et al. 1996, Genetics; 144(4), 1425-1436). P069-4a was further developed by the disruption of the ABC transporters PDR5 and SNQ2 . Strains with such double mutations pdr5 and snq2 exhibit a particularly high ZEN absorption (Mitterbauer et al, 2003, Appl. Environ. Microbiol.; 69(2), 805-811). This strain was subsequently transformed with an expression vector (pTK103) that triggers the production of a fusion protein containing the hormone-dependent activation domain of the human estrogen receptor aloha and the DNA binding domain of the yeast Gal4 protein, in order to obtain the strain YZHB817.

In the presence of estrogenic substances, the yeast strain YZHB817 induces the expression of the GAL7 lacZ reporter gene, whose product, the enzyme beta-galactosidase, is easy to measure by the hydrolyzation of the chromogenic substrate ONPG (ortho-nitrophenyl-beta-galactoside) at 420 nm (Current Protocols in Molecular Biology, Chapter 13, Yeast (Eds. Lundblad V, Struhl, K.)).

100 ml of the estrogenic test solution (ZEN or iZOM) were dissolved in 50% ethanol, mixed with 1.9 ml of the yeast culture of strain YZGA817 in SC-TRP medium at a cell density of OD₆₀₀=0.1, and incubated for 18 hours at 30° C. and 180 rpm. After this, the OD₆₀₀ of the culture was determined, and the cell pellet (10 min, 13 krpm) of 1 ml was resuspended in 500 ml Z buffer and permeabilized with 25 μl chloroform. The enzyme reaction was started by the addition of 100 μl ONPG solution (4 mg/ml) and stopped after yellow coloring upon addition of 250 μl of a 1M Na₂CO₃ solution. The supernatant was measured at 420 nm, and the relative units (based on the used cell amount) were calculated as:

RU=(OD ₄₂₀×1000)/(OD ₆₀₀×incubation time in min)

As illustrated in FIG. 4, ZEN induces the expression of the GAL7-lacZ reporter gene in the low ppb range, thus approximately 45 ppb ZEN are necessary to express 7.5 relative units of beta-galactosidase (FIG. 4A). By contrast, iZOM exhibits a far lower estrogenicity. In comparison, about 500 ppb were required to achieve approximately the same effect (FIG. 4B). Hence results that iZOM has an estrogenic activity approximately reduced by a factor 100 as compared to ZEN.

EXAMPLE 4 Determination of the Sequence Identity

The determination of the percentual sequence identity of the polypeptides with the amino acid sequences SEQ ID Nos. 1, 2 and 3 relative to one another was performed by the alignment of two sequences using the program BLAST (Basic Local Alignment Search Tool), in particular BLASTP, which can be used on the homepage of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). It is thereby possible to compare two or several sequences with one another according to the algorithm of Altschul et al., 1397 (Nucleic Acids Res. (1997) 25: 3389-3402). The base adjustments were used as program adjustments, but in particular: “max target sequence”=100; “expected threshold”=10; “word size”=3; “matrix”=BLOSOM62; “gap costs”=“Existence: 11; Extension: 1”; “computational adjustment”=“Conditional compositional score matrix adjustment”. The sequence identity between the sequences of SEQ ID No. 1 and SEQ ID No. 3 is 73%. 

1. A polypeptide for the enzymatic detoxification of zearalenone, wherein the polypeptide is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and that the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2 and 3 or a functional variant thereof, the functional variant and at least one of the amino acid sequences having a sequence identity of at least 60% to 90%.
 2. The polypeptide according to claim 1, wherein the monooxygenase is a Baeyer-Villiger monooxygenase.
 3. The polypeptide according to claim 1, wherein the monooxygenase is a cyclohexanone monooxygenase.
 4. The polypeptide according to claim 1, wherein the polypeptide, in particular monooxygenase, converts zearalenone to iZOM in a one-step enzymatic oxidation reaction so as to detoxify at least 70% of the zearalenone within 24 hours at 30° C. by converting the keto group in position 7 of the zearalenone into an ester group, said polypeptide being formed by a transformed yeast strain YZGA515, which is transformed with a pCS57 vector additionally containing a polynucleotide for expressing the polypeptide, and the transformed yeast strain is used at a cell density OD600 of 4, and zearalenone is used as a substrate at a concentration of 2 mg/l, and SC-LEU medium is used as a reaction medium.
 5. A transgenic host cell for preparing a zearalenone-detoxifying monoxygenase, wherein the host cell expresses a polynucleotide, that the polynucleotide comprises a nucleotide sequence encoding at least one polypeptide according to claim 1 and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, said polynucleotide being chromosomally integrated or extrachromosomally present and the host cell being a plant cell, and that optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively.
 6. Seed comprising a transgenic plant cell according to claim
 5. 7. An additive for the enzymatic detoxification of zearalenone, wherein said additive comprises at least one polypeptide according to claim 1 and at least one adjuvant selected from the group consisting of vitamins, minerals, enzymes, farther components for detoxifying mycotoxins and cofactors, in particular NADPH and/or NADH, enzyme preparations such as proteases, amylases, cellulases or glucanases, hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases and/or combinations thereof, mycotoxin-detoxifying enzymes such as aflatoxin oxidase, ergotamine hydrolases, ergotamine amidases, ochratoxin amidases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol aminoxidases, deoxymyalenol epoxide hydrolases; mycotoxin-detoxifying microorganisms; mycotoxin-binding components such as microbial cell walls, and inorganic materials such as bentonite.
 8. The use of an isolated polynucleotide encoding at least one polypeptide according to claim 1 and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5and 6 for preparing a zearalenone-detoxifying monooxygenase.
 9. The use of an isolated polynucleotide encoding at least one polypeptide according to claim 1 and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5and 6 in a method for preparing a zearalenone-detoxifying monooxygenase.
 10. A method for preparing a zearalenone-detoxifying monoxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to claim 1 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into a transgenic host cell or extrachromosomally provided, that the host cell is a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell.
 11. The use of an isolated polynucleotide encoding a zearalenone-detoxifying polypeptide for preparing a transgenic host cell, wherein the host cell expresses the isolated polynucleotide, which comprises a nucleotide sequence encoding at least one polypeptide according to claim 1 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, wherein the polynucleotide is chromosomally integrated or extrachromosomally provided and the host cell is a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, and that optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively.
 12. A method for preparing a transgenic host cell for preparing a zearalenone-detoxifying monooxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to claim 1 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host cell, and that, optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell.
 13. The use of a transgenic host cell for preparing a zearalenone-detoxifying monooxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to claim 1 and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell. 